Prevalence of binary-toxin genes (cdtA and cdtB) among clinical strains of Clostridium difficile isolated from diarrheal patients in Iran.

Aim
In this study we investigated the prevalence of binary toxin genes, cdtA and cdtB, in clinical isolates of C. difficile from hospitalized patients with diarrhea.


Background
C. difficile binary toxin (CDT) is an action-specific ADP-ribosyltransferase that is produced by some strains of C. difficile. Co-expression of this toxin with tcdA and tcdB can lead to more severe disease in CDI patients.


Methods
Totally, 930 patients suspected of having CDI was included in this study. All samples were treated with methanol and cultured on selective C. difficile agar plates. The C. difficile isolates were further identified by PCR. Presence of tcdA, tcdB, cdtA, and cdtB genes among the strains were examined by PCR.


Results
Analysis of the PCR results showed a prevalence of 85.2% (144/169) for toxigenic C. diffidile. Toxin genotyping of the strains for tcdA and tcdB genes revealed the toxin profiles of A+B+, A+B-, A-B+ accounting for 86.1% (124/144), 7.6% (11/144), 6.2% (9/144) among the strains, respectively. Totally, 12.4% (21/169) of the C. difficile strains were binary toxin-positive. cdtA-B+, cdtA+‏B‏+ and cdtA+B- were detected in 43% (9/21), 38% (8/21) and 19% (4/21) of the strains, respectively. Interestingly, 12% (3/25) of nontoxigenic C. difficile strains (tcdA-B-) had either cdtA+‏B‏+ or cdtA-B+ profiles.


Conclusion
This is the first report for the prevalence of binary toxin genes in C. difficile strains isolated from Iran. Further studies are required to investigate the exact role of binary toxins in the pathogenesis of C. difficile particularly in patients with chronic diarrhea among Iranian populations.


Introduction
1 Clostridium difficile is a gram-positive spore forming anaerobic bacterial pathogen. This microorganism is considered as the major causative agent for 5 to 25% of antibiotic associated diarrhea (AAD) and also pseudomembranous colitis (PMC) (1). Toxigenic strains generally produce two main toxins, including toxin A (enterotoxin) and B (cytotoxin), which have been identified as the main virulence factors in the pathogenesis of these bacteria (2,3). The genes encoding toxin A and B are clustered in a specific locus, the pathogenicity locus called PaLoc. This locus is composed of five genes, including tcdA and tcdB that encode the over-mentioned toxins, tcdE encodes a

ORIGINAL ARTICLE
putative holin for extracellular release of tcdA and tcdB, and tcdR and tcdC that encode the regulatory proteins (4). Popoff et al. discovered a binary toxin in the historic CD196 strain, called CDT in 1988 (5). This toxin is found in a few strains of C. difficile and consists of a binding component and an enzymatic component that displays an action-specific ADP ribosyltransferase activity that leads to cytoskeleton disorganization (6). The genes encoding these two components, cdtA and cdtB, and a regulatory protein, are co-located on a locus called CdT. This toxin might potentiate the toxicity of tcdA and tcdB and lead to more severe disease and could, thus, be considered to be an accessory virulence factor (7). It has been estimated that about 1.6 to 10% of C. difficile isolates carry the binary toxin genes in their genomes (8).
In spite of high prevalence of toxin-A-negative/toxin-B-positive C. difficile strains among hospitalized patients, there are several reports about involvement of tcdA/B-negative strains in the occurrence of AAD and enterocolitis (9)(10)(11)(12)(13). Although overgrowth of tcdA/Bnegative strains in the intestine and the absence of other enteric pathogens may support this involvement, further studies are required to determine the role of accessory virulence factors in the pathogenesis of C. difficile infection (CDI). Involvement of CDT in induction of apoptosis through its DNAse activity was shown in an in vitro study (14). This activity could mimic the same pathophysiological effects in the intestine, as was shown for tcdA/B toxins (8). Several clinical studies have suggested an association between the CDTencoding C. difficile strains and increased mortality of the patients (15), however its establishment needs further studies. In the current study we aimed to investigate the prevalence of binary toxin genes, cdtA and cdtB, in clinical isolates of C. difficile recovered from hospitalized patients with diarrhea.

Bacterial isolates
The study population consisted of 169 clinical isolates of C. difficile, which had been recovered from the fecal samples of 930 patients with chronic diarrhea referred to the Diagnostic Anaerobic Laboratory of Research Institute for Gastroenterology and Liver Diseases in Tehran, Iran. All demographic and clinical data of the patients, including age, gender, antibiotic treatment and medications history were collected by using a standard questionnaire.

Culture and isolation
All fecal samples from patients cultured on proper culture media after their treatments by the following methods. First, small amount of stool samples were mixed with 1 ml of 5% yeast extract broth and directly inoculated onto C. difficile medium (Mast, London, United Kingdom) supplemented with 7% horse blood and C. difficile selective supplement consisting of Dcycloserine (250 mg/ liter), cefoxitin (8 mg/liter), and lysozyme (5 mg/liter) (Mast, London, United Kingdom). The cultured plates were incubated at 37 °C for at least 48-72 h under anaerobic conditions (80% N 2 ; 10% CO 2 and 10% H 2 ) in an anaerobic generation system Anoxomat-Mart (Microbiology, Holland). Second, the samples treated with 1 ml of methanol (alcohol-shock procedure) for 1 to 2 minutes before inoculation on C. difficile medium (16,17). The cultures results were followed up to one week. Isolates with characteristic colony morphologies and Gram staining for C. difficile were further identified by PCR using specific primers (18). Subcultures of the confirmed isolates were stored in cooked meat broth (Himedia, India) at 4 ˚C.

Genomic DNA extraction
The genomic DNA was extracted from pure colonies of C. difficile grown on C. difficile medium agar plates. DNA was extracted by Instagene matrix extraction kit (Bio-Rad, Nazareth, Belgium) and boiling method (19). In the boiling method, a loop full of each colony was resolved in 500 µl of distilled water and homogenized and centrifuged for 10 min at 13000 g. The pellets were mixed with 100 µl of distilled water, vortexed and incubated for 10 min in water bath. The samples were then centrifuged at 13000 g for 8-10 min. The supernatant containing bacterial DNA was stored at -20 °C, until further use.

Detection of binary-toxin genes
For molecular identification of C. difficile isolates, PCR was done using specific primers for cdd3 gene of PaLoc. All primer pairs used in this study are presented in Table 1. PCR amplification was also done by using specific primers for tcdA and tcdB genes as previously described by Spigaglia et al. (18). For detection of cdtA and cdtB genes, PCR was done using specific primers (20) in a 25μl reaction mixtures containing 1X PCR buffer (50 mM KCl, 10 mM Tris-HCl), 1.5μM MgCl 2 , 0.3μM of cdtA and 0.5μM cdtB primers, and 1U of Taq DNA polymerase. PCR amplifications of 375 bp fragment of the cdtA was done in a thermocycler (Eppendorf, Hamburg, Germany) as follows: initial denaturation at 5 min at 95 °C, followed by 40 cycles of 1 min at 95 °C, 50s at 57.5 °C and 35s at 72 °C; and a final extension at 72 °C for 5 min to end amplification process. For amplification of 510 bp fragment of the cdtB the following time-temperature profile was used: 5 min at 95 °C for initial denaturation, 40 cycles of 1 min at 95 °C, 1 min at 58.9 °C, and 35 at 72 °C; and a final extension cycle of 10 min at 72 °C. Peptoclostridium difficile strain RIGLD141 was used as the control positive strain in amplification experiments. The partial nucleotide sequences for cdtB (KM047900.1) and cdtA-like (KM047901.1) genes were deposited in the GenBank/NCBI.

Statistical analysis
Statistical analyses were performed using IBM SPSS Statistics version 21 (Armonk, NY: IBM Corp.). Clinical and demographic data were analyzed by the Chi-square and Fisher's exact tests. A p value less than 0.05 was regarded as indicating a statistically significant difference.

Prevalence of CDI
This study investigated 930 suspected patients to CDI, including 466 males and 464 females. The CDI was confirmed in 169 patients (18.2%), 85 men and 84 women ranged between <1 to 50 years old. The prevalence of CDI varied among the patients in different wards of hospital and was as follow; 22.6% (38/169) in infectious diseases ward, 18.9% (32/169) in internal medicine ward, 12.4% (21/169) in intensive care unit (ICU), 9.5% (16/169) in surgical ward, and 4.8% (8/169) in gastroenterology ward. The lowest prevalence was detected among the patients in bone marrow and kidney transplantation units with less than 5%. Most of the CDI patients presented a defecation rate of 3 times per day, which was higher than CDInegative patients with ≤2 times per day.

Frequency of tcdA, tcdB and binary toxins among the C. difficile strains
Of total patients with CDI, toxigenic and nontoxigenic C. difficile strains were characterized in 85.2% (144/169) and 14.8% (25/169) of the patients, respectively. Toxin genotyping of the strains for tcdA and tcdB genes revealed the toxin profiles of A + B + , A + B -, A -B + accounting for 86.1% (124/144), 7.6% (11/144), 6.2% (9/144) among the strains, respectively. Nearly, half of the toxigenic C. difficile strains (51.7%) with toxin profile of tcdA + B + were detected among the patients who had 3-5 defecations per day. More than half of the patients (63.6%) with toxin profile of tcdA + B -were seen among the males, while most of the females (66.7%) had toxin profile of tcdA -B + . Among all toxigenic patients, cancer was observed as the most common underlying disease. The highest (26.3%) prevalence of toxigenic C. difficile strains was observed in patients confined in infectious diseases ward, and the lowest (0.7%) was seen in coronary care unit (CCU), endocrinology and orthopedic wards. Totally, 12.4% (21/169) of the C. difficile strains were binary toxin-  Table 2.

Distribution of tcdAB genes in relation to binary toxin profiles
Most of the C. difficile strains with cdtA + B + and cdtA -B + profiles were found to be tcdA + B + . Moreover, half of the strains with cdtA + B -profile were found to be tcdA + B + . Interestingly, 12% (3/25) of nontoxigenic C. difficile strains (tcdA -B -) were found to have either cdtA + B + or cdtA -B + profiles. None of the strains with cdtA + B + and cdtA -B + profiles were detected to be tcdA -B + . In addition, strains with cdtA + B -profile carried at least one of the tcdA or/and tcdB genes. Distribution of tcdAB toxin genes in relation to cdtAB binary toxin profiles were shown in Table 3.

Discussion
The structure of the C. difficile binary toxin is well known, but few data are available to show the role of this toxin in the pathogenesis of gastrointestinal disease. Currently, there is little information about the clinical relevance and pathogenic role of the ADPribosylating toxin CDT in C. difficile infections (21). C.
difficile induces a wide range of diseases from mild diarrhea to PMC, toxic megacolon, and even fulminant colitis. It has been reported that variations in the expression levels of tcdA and tcdB as the major C.
difficile toxins cannot account for the wide spectrum of clinical presentations (7,21). Borriello et al. reported no correlation between virulence in a hamster model of antibiotic association colitis and the production of tcdA and tcdB in vitro (22). Therefore, it was hypothesized that CDT produced by some C. difficile strains may be responsible for these manifestations. CDT is a potent cytotoxin, which impairs the functions of mucosal barrier and subsequently can facilitate the action of typical Clostridial cytotoxins (23). CDT may also act in synergy with other toxins, depolymerizing the actin cytoskeleton by a complementary mechanism (24). In C. difficile strain CD196 that can cause a severe PMC, the production of this additional toxin exacerbates the symptoms of PMC (23). In the present study, C. difficile in humans and animals (30)(31)(32)(33), the prevalence of this toxin type has been reported to be less than 5% in humans (34). Binary toxin producing strains of C.
difficile has been reported in recent years, and the prevalence of binary toxin genes vary in different geographic regions worldwide. The deduced prevalence of binary toxin genes in toxigenic strains is high and differences in the findings of prevalence reports may be partly due to the differences in patient (symptomatic or not, adult or children) or strain selection. In addition, as was shown recently by Rupnik (37). In our study, the frequency of A + B + CDT + , A + B + CDT -andA -B + CDTwere 6/169 (3.6%), 110/169 (65%), and 8/169 (4.7%), respectively. In a study conducted in Japan the prevalence of A + B + CDT + , A + B + CDTand A -B + CDTwere 4/71(5.6%), 58/71(81.7%), and 9/71 (12.7%), respectively (38,39). In conclusion, to our knowledge this is the first report for the prevalence of binary toxin genes in C. difficile strains isolated from Iran. Further studies are required to investigate the exact role of binary toxins in the pathogenesis of C. difficile particularly in patients with chronic diarrhea among Iranian populations.